Migraine is the most common neurological disorder, the 3rd most common disease on earth, and the 8th most disabling. Currently available treatments fail to effectively manage migraine in most patients. Development of new therapeutics has been slow due in large part to a poor understanding of the underlying pathology of migraine. Endogenous proteases, released in the meninges by resident mast cells, have been proposed as a potential driver of migraine pain via an action on protease activated receptor type 2 (PAR2). Unfortunately the evidence for this mechanism relies on imprecise pharmacological tools and lacks genetic validation. The central hypothesis of this multi-PI research program is that PAR2 expression in nociceptors that project to the meninges plays a key role in the pathogenesis of migraine pain by linking meningeal protease release to sensitization of the trigeminal nociceptive system. The primary objectives of this proposal are to develop next generation PAR2 antagonists, to use these tools to validate PAR2 as a migraine pain target and to use mouse genetics to identify the specific role of PAR2 expression in meningeal projecting nociceptors to migraine pain. Our first aim will be to use our established PAR2 development pipeline to design new PAR2 antagonists with improved drug-like properties to probe PAR2 function in the context of a mouse model of migraine pain. Our second aim will use pharmacological tools in a novel mouse migraine model to further understand the potential role of PAR2 in migraine and signaling pathways engaged by PAR2 to evoke pain from the dura. Our final aim will use mouse genetics to study the cell type-specific role of PAR2 in migraine pain. Our work will result in: 1) the discovery and development of novel high potency antagonists for PAR2; 2) the development of an innovative new target for migraine pain; and 3) provide the first genetic verification of the cell type-specific action of PAR2 in the pain pathway. Taken together, successful studies will provide a preclinical rationale for the further development and testing of PAR2 ligands for the treatment of migraine and other forms of pain.